One of the major challenges in biochemistry is to understand the functional genomics of organisms. It is a staggering problem when one considers the fact that about 40% of the genes in of one of the best-understood organisms, Escherichia coli, are not experimentally defined. The organism colonizes the human intestine shortly after birth and is, along with its close relatives such as Salmonella, Shigella and Yersinia, a pathogen that is a significant source of human disease. Glutathione (GSH) is the predominant redox active thiol in most aerobic organisms where it plays a fundamental role in metabolic, catabolic and redox chemistry. GSH transferases are enzymes that participate in this chemistry by adding GSH to electrophilic acceptors. The E. coli genome harbors genes encoding nine glutathione (GSH) transferase homologues. Amazingly, only one gene has a reasonably well-defined function and it does NOT encode a GSH transferase but rather stringent starvation protein A, SspA, a transcription factor. Under aerobic conditions, GSH is the predominate thiol in E. coli. However it is largely converted to glutathionylspermidine (GspSH) under anaerobic conditions by glutathionylspermidine synthetase/amidase (GSS). The broad, long-term objectives of this project are to understand the GSH/GspSH homeostasis in E. coli and the biological activities and roles of chromosomally encoded GSH transferase homologues in E. coli. Ten interrelated proteins are the subject of this investigation. They include the eight canonical GSH transferase homologues, YliJ, YncG, Gst, YfcF, YfcG, YghU, SspA, and YibF as well as GSS, and the membrane-bound GSH transferase, YecN. The research plan includes three specific aims. The first aim is to understand the mechanism and regulation of GSH/GspSH homeostasis in E. coli. The second aim is to define the biological functions of the eight canonical GSH transferase homologues in E. coli. The third aim is to determine the biological role of the single membrane-bound GSH transferase homologue. These aims will be achieved by a multidisciplinary approach that includes: (i) analysis of genome context;(ii) phenotypic responses to gene knockouts;(iii) response of gene expression to growth conditions and stress;(iv) a search for protein partners;(v) determination of X-ray crystal structures of the proteins;(vi) functional and kinetic characterizations of the proteins. PUBLIC HEALTH RELEVANCE: Escherichia coli is a member of the Enterobacteriaceae family that includes several intestinal pathogens such as Salmonella, Shigella and Yersinia. The bacterium typically colonizes the human intestine shortly after birth where it remains the predominant facultative microorganism. Understanding the biochemistry of E. coli is fundamental to understanding its pathogenesis.